Cellular osmotic homeostasis is a fundamental requirement for life. All cells are exposed to osmotic challenges brought about by changes in intracellular solute flux and/or perturbations in extracellular osmolality. Most mammalian cells are protected from extracellular osmotic challenges by the kidney, which tightly regulates blood ionic and osmotic concentrations. Renal medullary cells are an important exception to this generalization and are subjected normally to extreme osmotic stress by the renal concentrating mechanism. Cells maintain osmotic homeostasis by the tightly regulated gain and loss of salt and organic solutes termed organic osmolytes, and by detecting and repairing osmotic stress induced damage. The transport and metabolic pathways that mediate animal cell osmoregulatory solute fluxes are well described. However, little is known about the signaling mechanisms by which animal cells detect osmotic perturbations, about the types of cellular and molecular damage induced by osmotic stress, and about how this damage is detected, repaired and prevented. DK61168 supported studies developed the nematode C. elegans as a novel genetically tractable model system for defining fundamental mechanisms of animal cell osmosensing and osmotic homeostasis. During the previous funding period, we made the novel observation that disruption of protein synthesis activates expression of genes required for organic osmolyte accumulation. We also demonstrated for the first time that hypertonicity causes rapid and extensive protein damage in vivo and that genes required for protein degradation are essential for survival during hypertonic stress. The current proposal builds on these new findings and addresses three questions with broad biological and pathophysiological significance. How does disruption of protein synthesis activate osmosensitive gene expression? What are the quality control mechanisms utilized by cells to detect, degrade and repair proteins damaged by hypertonic stress? What are the mechanisms by which acclimation to hypertonic stress suppresses hypertonicity induced protein damage? We will utilize a combination of cell biological, molecular and biochemical approaches to provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. We will also exploit the genetic tractability of C. elegans and begin to define the signals and signaling pathways that regulate expression of genes required for survival in hypertonic environments. Our work will provide novel insights into cellular osmosensing and signal transduction and into the mechanisms that protect hypertonically stressed cells from protein damage and associated injury and death. Detailed understanding of hypertonicity induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases. Public Health Relevance: Cellular osmotic homeostasis is a fundamental requirement for life. Studies described in this application will define the signals and signaling pathways that regulate expression of genes required for survival of cells in hypertonic environments and will provide the first detailed characterization of hypertonic stress induced protein damage and the mechanisms that cells employ to cope with and prevent this damage. Detailed understanding of osmotic stress induced signaling, cell injury and protein damage is essential for understanding renal physiology and pathophysiology, and is directly relevant to understanding pathophysiology associated with aging and numerous inherited diseases.